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Pharmacalogical characteristics of adenosine A1-receptor-stimulated CRE-gene transcription
It has previously been shown that the human adenosine A1-receptor can couple to multiple heterotrimeric G-proteins (Cordeaux et al., 2000). Previous reporter gene studies of the Gs-coupled β2-adrenoceptor have indicated that estimates of antagonist affinity vary depending on the efficacy of the agonist used to stimulate the receptor (Baker et al., 2003). Here, we used a cyclic AMP response element (CRE) based reporter gene response to explore the pharmacological characteristics of human A1 adenosine receptors. CHO cells stably transfected with a CRE-SPAP (secreted placental alkaline phosphatase) reporter gene and the human A1 receptor were used. Measurements of 3H-cAMP and CRE-SPAP production were made as previously described (Baker et al., 2003). Cyclopentyladenosine (CPA) inhibited forskolin-stimulated 3H-cAMP accumulation (log IC50= -9.17±0.09, n=7). However, at higher CPA concentrations, a small increase in 3H-cAMP accumulation was also seen (log EC50= -6.19±0.06, n=6). 24hr pre-incubation with pertussis toxin (100ng/ml, PTX, pertussis toxin) abolished the inhibitory component, however the stimulatory response remained (log EC50 = -6.82±0.03, n=8). A similar biphasic concentration-response was observed for the CRE-SPAP response (log IC50= -8.46±0.06, log EC50 = -6.42±0.07, n=10), however the stimulatory part of the response was more easily seen. Again the inhibitory response was abolished by PTX, but the stimulatory response remained (log EC50 = -6.42±0.07, n=10). It is therefore likely that the PTX-insensitive stimulatory responses seen at high agonist concentrations are due to A1-receptor coupling to Gs-proteins. Similar biphasic responses were seen with other agonists – NECA (adenosine-5’-N-ethyluronamide), GR79236, 2-chloroadenosine, 2-chloro-CPA and phenylisopropyladenosine (PIA). DPCPX (8-cyclopentyl-1,3-dipropylxanthine) antagonised both the inhibitory and stimulatory components of these responses to yield similar log KD values regardless of which agonist was present (see Table). Furthermore, the Schild slopes for antagonism by DPCPX at both conformations of the A1-receptor were never different from 1 suggesting all interactions were competitive. In conclusion, a range of agonists can stimulate the adenosine A1 receptor to couple to both Gi and Gs proteins. The affinity of antagonists to both of these conformations are the same and unlike the β2-adrenoceptor do not vary with the competing agonist. Log KD values for DPCPX with a range of A1-agonists at both the PTX-sensitive inhibitory Gi-conformation and the PTX-insensitive stimulatory Gs-responses. Cordeaux Y. et al., (2000) Mol. Pharmacol. 58, 1075-1084. |
